123count ebeads

Spleens and lymph nodes (cervical, brachial, axillary and inguinal) were harvested, smashed using 120μM nylon sheets, filtered through a 70μM filter, and subjected to ACK lysis. Tumors were harvested, cut into 2 mm pieces and processed using the gentleMACS Dissociator according to the manufacturer's instructions (Miltenyi Biotec). After digestion, single cell suspensions were filtered through a 70μM filter. TILs were enriched using a 44%/67% Percoll (GE Healthcare) gradient. All cell counts were performed using 123count eBeads (eBioscience). Approximately 1–10 × 10⁶ immune cells were stained for flow cytometry.

Single-cell kidney and peritoneal suspensions were blocked with 0.5% heat-inactivated mouse serum, followed by extracellular staining for 1 h at 4 °C with a combination of antibodies listed in the SI Appendix. Viability staining was performed with Zombie UV Fixable Viability Kit (BioLegend) for 20 min at room temperature. For intracellular cytokine staining, cells were fixed and permeabilized using the Cytofix/Cytoperm kit (BD Bioscience) as per the manufacturer’s instruction. Staining was carried out for 1 h at room temperature using pro-IL-1β (NJTEN3; eBioscience) at a 1:200 dilution. Cell counting was performed using 123count eBeads (eBioscience). Flow cytometry data collection was performed on an LSR Fortessa flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Treestar, v10.2).

Flow cytometric analysis of kidney and bladder infiltrates was performed on single cell suspensions isolated following digestion. Following infection, kidneys and bladders were homogenized in RPMI media (Thermo Fisher Scientific, Waltham, MA) containing 1 mg/ml Collagenase D and 100 μg/ml DNase I (Roche Diagnostics, Indianapolis, IN), supplemented with 20 mM HEPES and 10% fetal calf serum (FCS). Suspensions were and digested for 45 min at 37 °C, passed through 40 μm cell strainers, treated with BD Pharm Lyse™ (BD Biosciences, San Diego, CA), and cell pellets rinsed in PBS with 2% FCS. For cell staining 1 × 10⁶ cells were placed on ice in 12 × 75 mm tubes, blocked with Fc Block™ and stained with a cocktail of conjugated antibodies for 15 min, rinsed in PBS with 2% FCS, and stained with 3 μM DAPI (4′,6-diamidino-2-phenylindole, Thermo Fisher Scientific, Waltham, MA). Absolute cell numbers were determined using 123count eBeads (eBioscience, San Diego, CA) following the manufacturer’s instructions. Samples were analyzed with a BD LSR II flow cytometer and FACSDiva software (BD Biosciences, San Diego, CA). Antibodies used included: BV605 rat anti-mouse CD45, PerCP-Cy5.5 rat anti-mouse Ly-6C, FITC rat anti-mouse Ly-6G (BD Biosciences, San Diego, CA), and APC anti-mouse CD11c, PE/Cy7 anti-mouse CD11b, and PE anti-mouse F4/80 (Biolegend, San Diego, CA).

For analysis of cytokine production, cells were restimulated with 1 μg/mL ionomycin and 50 ng/mL PMA in the presence of 5 μg/mL brefeldin A for 4 h. Cell surface staining was carried out in PBS with 2% FBS. For intracellular staining, cells were first stained with Zombie Aqua Fixable Viability kit (Biolegend), followed by staining for cell-surface markers and then resuspended in fixation/permeabilization solution (3% BSA and 0.5% saponin in PBS). Samples including the analysis of Foxp3 transcription factor were resuspended in fixation/permeabilization solution (Foxp3 Fixation/Permeabilization; eBioscience) according to the manufacturer instructions. To determine absolute number of cells, samples were analyzed in the presence of 123count eBeads (eBioscience) according to manufacturer’s guidelines. Data were collected with a Canto II (BD) and results were analyzed with FACSDiva (BD) and FlowJo software (Tree Star, Ashlan, OR, USA).